Method of stabilizing dye solutions and stabilized dye compositions

ABSTRACT

Stabilized metachromatic dyes, especially at application concentrations, comprising metachromatic dyes dissolved in one or more non-aqueous solvents, as well as metachromatic dyes dissolved in pH stabilized aqueous solutions. Also, various combinations of treatments are disclosed for stabilizing metachromatic dyes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/441,662,filed Nov. 16, 1999 now U.S. Pat. No. 6,241,788. The entire disclosureof application Ser. No. 09/441,662 is considered as being part of thedisclosure of this application, and the entire disclosure of applicationSer. No. 09/441,662 is expressly incorporated by reference herein in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the stabilization of metachromaticdyes, and compositions comprising stabilized metachromatic dyes. Thepresent invention is also directed to processes for determiningpolyionic materials utilizing stabilized metachromatic dye compositions.

2. Discussion of Background Information

Colorimetry is a well-known method of chemical analysis which involvesthe comparison and matching of a standard color with that of an unknowncolor to approximate the concentration of a specific component in asample to be analyzed. When the amount of light absorbed by a givensubstance in solution is proportional to the concentration of theabsorbing species, colorimetry is a simple and accurate method fordetermining unknown concentrations. For example, if the concentration ofa polymer in an aqueous system is to be determined, a sample can betaken, the absorbance of the sample in the presence of a suitable dyecan then be measured and compared with a calibration curve to quicklyand accurately estimate the concentration of the polymer in the aqueoussystem. Colorimetry provides advantageous testing since it can easily beperformed at the application site.

Certain dyes undergo a unique color change upon interaction withpolyionic compounds in solution known as metachromasy. Thus,metachromatic dyes are those which undergo a color change uponinteraction with polyionic compounds. Any metachromatic dye can be usedin a calorimetric test to determine the concentration of a substance,including polycarboxylates, sulfonates, and the like in an aqueoussolution. More specifically, when anionic polymers contact ametachromatic dye, the dye molecules align with the anionic charges onthe polymers, resulting in a shift in the wavelength of maximumabsorbance of the dye molecule. This shift is observable as a colorchange in the solution containing the dye and the polymer. Thus, sincepolycarboxylates and sulfonates, which are anionic, induce ametachromatic change in certain dyes, their concentrations in aqueoussolutions can be determined calorimetrically by measuring theabsorbance, at a specified wavelength, of a solution containingpolycarboxylates and/or sulfonates and a metachromatic dye and comparingthis absorbance to absorbances of standards having known concentrationsof the species being measured.

However, when metachromatic dyes are dissolved in aqueous solutions foruse in analytical determinations, fresh samples must be prepared on adaily basis to insure accurate analysis. Aqueous solutions ofmetachromatic dyes are extremely susceptible to degradation due to avariety of factors, such as light, temperature, dissolved oxygen, pH,etc. For example, when pinacyanol chloride is dissolved in an aqueoussolution at a concentration of 9.0×10⁻⁵ molar, the pinacyanol chloridedegrades at a rate of approximately 10 to 20% per week. Because of thisdegradation problem, frequent reagent preparations must be made in thefield and this is not practical.

Still further, the instability of known dye solutions leads todisadvantageous results associated with the detection process. Thesedisadvantages result in a lack of reproducibility of results, i.e.,consistency of results is difficult to attain on separate days even withthe same water sample.

Accordingly, there is a need to provide stable metachromatic dyes thatenable simple tests for materials, particularly water treatmentpolymers, and especially enable simple tests that can be utilized overextended periods of time without the need for formulation in the field.

SUMMARY OF THE INVENTION

The present invention concerns methods for stabilizing metachromaticdyes so that the dyes will not be susceptible to degradation andstabilized compositions comprising the metachromatic dyes. This willensure that any colorimetry testing using the stabilized dyes can beperformed with the added assurance of reproducible results.

The present invention is directed to stabilized metachromatic dyecompositions, including aqueous solutions and non-aqueous solutions ofmetachromatic dye, having a percent change in absorbance of less thanabout 10% when stored for a period of about one week, more preferablyless than about 1% when stored for a period of about one week, even morepreferably less than about 3% when stored for a period of about onemonth, even more preferably less than about 5% when stored for a periodof about 3 months, even more preferably less than about 5% when storedfor a period of about 6 months, even more preferably less than about 10%when stored for a period of about one year, and even more preferablyless than about 5% when stored for a period of about one year.

Further, the present invention is directed to aqueous solutions ofmetachromatic dye comprising metachromatic dye in an aqueous solvent,the aqueous solvent having a metachromatic dye stabilizing pH.

Still further, the present invention is directed to non-aqueoussolutions of metachromatic dye comprising metachromatic dye andnon-aqueous solvent, the non-aqueous solution being substantially freeof water.

Still further, the present invention is directed to a stabilizedmetachromatic dye composition for analytical determination of at leastone polyionic substance, the metachromatic dye composition including aconcentration of metachromatic dye which provides maximum metachromaticabsorbance for the at least one polyionic substance when the at leastone polyionic substance is present at a concentration of 0. 1 to 1.5ppm, and the metachromatic dye composition has a percent change inabsorbance of less than about 10% when stored for a period of about oneweek.

Still further, the present invention is directed to a stabilized aqueoussolution of metachromatic dye comprising metachromatic dye in an aqueoussolvent, the aqueous solution having a metachromatic dye stabilizing pH,and a percent change in absorbance of less than about 10% when storedfor a period of about one week.

Still further, the present invention is directed to a metachromatic dyesolution for analytical determination of at least one polyionicsubstance comprising metachromatic dye and non-aqueous solvent, thesolution being substantially free of water, said metachromatic dyesolution including a concentration of said metachromatic dye whichprovides maximum metachromatic absorbance for the at least one polyionicsubstance when said at least one polyionic substance is present at aconcentration of 0.1 to 1.5 ppm, and said non-aqueous solvent comprisesat least one of methanol, ethanol, butanol, isopropanol, propanol,ethylene glycol, methylcellosolve, hexane, pentane, heptane, toluene,xylene, benzene, dichlorobenzene, acetone, ethyl acetate, diethyl ether,acetonitrile and dimethylsulfoxide.

Still further, the present; invention is directed to a process foranalytical determination of at least one polyionic substance in asample, comprising forming a mixture by mixing a metachromatic dyesolution and the sample, the metachromatic dye solution comprisingmetachromatic dye and non-aqueous solvent, the solution beingsubstantially free of water, and performing an absorbance measurement onthe mixture.

Still further, the present invention is directed to a process forproducing a stabilized aqueous solution of metachromatic dye, comprisingforming a solution of metachromatic dye in aqueous solvent, themetachromatic dye comprising pinacyanol chloride, and the solutionhaving a metachromatic dye stabilizing pH, and the stabilized aqueoussolution of metachromic dye having a percent change in absorbance ofless than about 10% when stored for a period of about one week.

Still further, the present invention is directed to a process forproducing a metachromatic dye solution for analytical determination ofat least one polyionic substance, comprising combining metachromatic dyeand non-aqueous solvent so as to form a metachromatic dye solution whichis substantially free of water, the metachromatic dye comprisingpinacyanol chloride, the metachromatic dye solution including aconcentration of the metachromatic dye which provides maximummetachromatic absorbance for the at least one polyionic substance whenthe at least one polyionic substance is present at a concentration of0.1 to 1.5 ppm, and the non-aqueous solvent comprises at least one ofmethanol, ethanol, butanol, isopropanol, propanol, ethylene glycol,methylcellosolve, hexane, pentane, heptane, toluene, xylene, benzene,dichlorobenzene, acetone, ethyl acetate, diethyl ether, acetonitrile anddimethylsulfoxide.

Still further, the present invention is directed to a process forproducing a metachromatic dye solution, comprising combiningmetachromatic dye and non-aqueous solvent so as to form a metachromaticdye solution which is substantially free of water, the metachromatic dyecomprising pinacyanol chloride, the non-aqueous solvent having a densityat 25° C. of about 0.95 to 1.2 g/cm³ and comprising a mixture ofmethanol and ethylene glycol.

The aqueous solution of metachromatic dye can comprise aqueous solventshaving a pH of at least about 8, more preferably a pH of at least about10, and even more preferably a pH of at least about 11. Preferred pHranges of the aqueous solvents include a pH range of about 8 to 14, evenmore preferably a pH range of about 11 to 12, and even more preferably apH range of about 11 to 11.5. The aqueous solution of metachromatic dyecan include at least one basic material, such as a buffer, or a materialsuch as at least one of sodium hydroxide, potassium hydroxide andlithium hydroxide, preferably sodium hydroxide. Moreover, the aqueoussolution of metachromatic dye can include at least one non-aqueoussolvent.

The metachromatic dye can comprise at least one of pinacyanol chloride,crystal violet, methyl green, malachite green, acridin orange,paraosaniline, nile blue A, neutral red, safrin O, methylene blue,methyl red, brilliant green, toluidine blue, new methylene blue,quinalizarin, tetrachrome, brilliant blue G, and mordant black II, andis preferably pinacyanol chloride.

The non-aqueous solvent can comprise at least one of alcohols,methylcellosolve, hexane, pentane, heptane, toluene, xylene, benzene,dichlorobenzene, acetone, ethyl acetate, diethyl ether, acetonitrile,dimethylsulfoxide, preferably at least one of methanol, ethanol,butanol, isopropanol, propanol and ethylene glycol.

The non-aqueous solution of metachromatic dye can include one or morenon-aqueous solvents, preferably a mixture of methanol and ethyleneglycol having a preferred 25 vol % of methanol to 75 vol % of ethyleneglycol, with the non-aqueous solution preferably having a density at 25°C. of about 0.95 to 1.2 g/cm³, more preferably about 1 to 1.1 g/cm³, andeven more preferably about 1 to 1.05 g/cm³.

The non-aqueous solution of metachromatic dye is preferably free ofwater.

The non-aqueous solvent preferably comprises high purity solvent.

The solutions of metachromatic dye preferably include at least oneoxygen scavenger.

The solutions of metachromatic dye preferably are in the substantialabsence of oxygen.

The solutions of metachromatic dye preferably are purged with an inertgas.

The present invention is also directed to containers including thereinthe solutions of metachromatic dye according to the present invention.The container at least reduces the transmission of light, and preferablyprevents the transmission of light.

The present invention is also directed to methods of determiningmaterials, such as polyionic materials, preferably polyionic polymersutilizing the metachromatic dye solutions according to the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present inventionwill be made apparent from the following description of the preferredembodiments, given as non-limiting examples, with reference to theaccompanying drawings, in which:

FIG. 1 depicts a calibration curve for HPS-I at 480 nm plottingabsorbance vs. HPS-I concentration; and

FIG. 2 depicts plots of absorbance vs. concentration for HPS-I plottingabsorbance vs. HPS-I concentration.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The particulars shown herein are by way of example and for purposes ofillustrative discussion of embodiments of the present invention only andare presented in the cause of providing what is believed to be the mostuseful and readily understood description of the principles andconceptual aspects of the present invention. In this regard, no attemptis made to show structural details of the present invention in moredetail than is necessary for the fundamental understanding of thepresent invention, the description making apparent to those skilled inthe art how varying forms of the present invention may be embodied inpractice.

Unless otherwise stated, all percentages, parts, ratios, etc., are byweight.

Unless otherwise stated, a reference to a compound or component includesthe compound or component by itself, as well as in combination withother compounds or components, such as mixtures of compounds.

Further, when an amount, concentration, or other value or parameter, isgiven as a list of upper preferable values and lower preferable values,this is to be understood as specifically disclosing all ranges formedfrom any pair of an upper preferred value and a lower preferred value,regardless whether ranges are separately disclosed.

The present invention is directed to stable compositions ofmetachromatic dyes. According to the present invention, themetachromatic dye composition is stable if the absorbance ofmetachromatic dye composition changes by less than about 10%, morepreferably less than about 5%, even more preferably less than about 3%,and most preferably less than about 1%, when stored for a period ofabout 1 week, more preferably about 1 month, even more preferably about3 months, even more preferably about 6 months, even more preferablyabout 7.5 months, even more preferably about 10 months, and even morepreferably about 1 year or more.

In order to determine the change in absorbance of the metachromatic dyecomposition, an initial absorbance of the metachromatic dye compositionis determined 3 minutes after mixing of the metachromatic dye and thesolvent at any wavelength in the visible spectrum of 300 to 700 nm toobtain the absorbance data for the initially prepared metachromatic dyecomposition. While one or more wavelengths can be utilized and/ormeasurements can be made over the whole spectrum, preferably thewavelength of light is preferably at or about the wavelength thatprovides maximum absorbance. The metachromatic dye composition, i.e.,either the same dye composition that was tested to provide the initialabsorbance, another portion of the same metachromatic dye composition,or a metachromatic dye composition that has been formulated to beidentical to the metachromatic dye composition tested for absorbance isthen stored at ambient temperature, i.e., at 25° C., for the period oftime under dark conditions, such as by being stored in an ambercontainer, for which the stability test is to be performed. Absorbanceof the metachromatic dye composition is determined at the samewavelength or wavelengths of light as for the initially preparedmetachromatic composition. Calculations are then performed to determinethe percent change in absorbance.

Still further, the stabilized metachromatic dye compositions accordingto the present invention are capable of use as metachromatic dyes inanalytic determination of polyionic compounds, particularly polyioniccompounds including polycarboxylate and/or sulfonate concentrations inaqueous systems.

The present invention is directed to any technique for maintaining thestability of the metachromatic dye composition, and a variety oftechniques for providing stability to metachromatic dye compositions aredisclosed herein. Furthermore, it is noted that stability techniquesdisclosed herewith can be utilized individually and in combination witheach other.

The present invention is directed to any metachromatic dye that can bestabilized according to the present invention. In particular,metachromatic dyes that are most preferred for the present inventioninclude metachromatic dyes that are suitable for use in calorimetrictests for determining polyionic compounds in aqueous systems, preferablyfor determining polycarboxylate and/or sulfonate concentrations inaqueous systems. Examples of metachromatic dyes include, but are notlimited to, pinacyanol chloride, crystal violet, methyl green, malachitegreen, acridin orange, paraosaniline, nile blue A, neutral red, safrinO, methylene blue, methyl red, brilliant green, toluidine blue, newmethylene blue, quinalizarin, tetrachrome, brilliant blue G, and mordantblack II, preferably nile blue A and/or pinacyanol chloride.

The stability of the metachromatic dye composition can be enhanced byany technique that provides stability of the metachromatic dyecomposition. The following embodiments of the present invention aretherefore to be considered non-limiting embodiments setting forthpreferred manners of stabilizing metachromatic dye composition, andproviding guidelines in order that one having ordinary skill in the artcan provide, without undue experimentation, techniques for providingstabilized metachromatic dye compositions according to the presentinvention.

With the above in mind, it is noted that in one aspect the presentinvention achieves stabilized aqueous metachromatic dye compositions byproviding an aqueous composition of the dye having a pH of at leastabout 8, more preferably at least about 10, and even more preferably atleast about 11, with preferred pH ranges being from about 8 to 14, morepreferably from about 10 to 13, even more preferably from about 10.5 to12.5, even more preferably about 11 to 12, and even more preferablyabout 11 to 11.5, with one preferred pH value being about 11. For easeof reference the pH or pH range at which the metachromatic dye isstabilized will be referred to herein as the metachromatic dyestabilizing pH. Thus, the terminology metachromatic dye stabilizing pHwill be utilized herein to denote a metachromatic dye composition havinga stabilizing pH in contrast to a non-stabilizing pH, such as an acidicpH.

The pH of the metachromatic dye composition can be adjusted and/ormaintained in various manners to provide the metachromatic dyestabilizing pH without effecting or substantially effecting the coloringchanging or substantially changing the coloring changing ability of themetachromatic dye and the metachromatic dye composition, and preferablywithout changing or substantially changing the absorbance of themetachromatic dye and metachromatic dye composition. For example, basicmaterials including, but not limited to, at least one of sodiumhydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide,calcium hydroxide, barium hydroxide, magnesium hydroxide and ammonia.Preferably the basic material is at least one of sodium hydroxide, suchas a 1N solution of sodium hydroxide, lithium hydroxide and/or potassiumhydroxide.

Additionally, the pH of the metachromatic dye composition can beadjusted and/or maintained using buffer systems that provide themetachromatic dye stabilizing pH without affecting or substantiallyaffecting the coloring changing or substantially changing the coloringchanging ability of the metachromatic dye and the metachromatic dyecomposition, and preferably without changing or substantially changingthe absorbance of the metachromatic dye and metachromatic dyecomposition. Preferably, the buffer system maintains a pH of about 9 to11 in the metachromatic dye composition. For example, buffer systemsinclude, but are not limited to, potassium carbonate/potassiumborate/potassium hydroxide (pH=10), boric acid/potassium chloride/sodiumhydroxide (pH=9), sodium hydroxide/glycine/sodium chloride (pH=11),sodium tetraborate (pH=9.18), and tris(hydroxymethyl)amino methane(pH=10.4).

The aqueous metachromatic dye compositions of the present invention caninclude other solvents therein in addition to water, such as non-aqueoussolvents. For examples, other solvents in addition to water can beincluded in the aqueous metachromatic dye compositions, such as, but notlimited to, ethanol, methanol, butanol, propanol, ethylene glycol,methyl cellosolve, and glycol. For example, the volume ratio of water toother solvent can range from about 99 vol % to 1 vol %, more preferablyfrom about 80 vol % to 20 vol %, even more preferably about 75 vol % to25 vol %, even more preferably about 50 vol % to 50 vol %, even morepreferably from about 20 vol % to 80 vol %, and even more preferablyfrom about 10 vol % to 90 vol %, and can even be as low as 1 vol % to 99vol %.

When the aqueous metachromatic dye composition is utilized in a test thepH of aqueous metachromatic dye composition is preferably adjusted to beat a neutral pH of about 6.5 to 7. Adjustment of the pH can be affectedin any manner that lowers the pH while not interfering with themetachromatic dye, such as by adding an acid, such as, but not limitedto, sulfuric acid, nitric acid, or a buffer, such as, but not limitedto, meta buffer, i.e., 10.1 wt % EDTA (ethylendiaminetetraacetic acid)tetrasodium salt, 11 wt % potassium phosphate monobasic, and 78.9 wt %distilled water.

In another aspect, the present invention achieves stabilizedmetachromatic dye compositions by providing non-aqueous compositions ofthe dye. In this regard, it has been discovered that the stability ofmetachromatic dyes is deteriorated by its inclusion in an aqueousenvironment. In particular, it is noted that when metachromatic dyes arestored in non-aqueous compositions stable metachromatic dye compositionsare obtained.

The non-aqueous metachromatic dye compositions according to the presentinvention can include water therein, such as trace amounts of water thatdo not affect the stability of the metachromatic dye compositions. Forease of reference, these compositions are referred to herein asnon-aqueous metachromatic dye compositions or solutions. In other words,the non-aqueous metachromatic dye compositions of the present inventioncan include amounts of water therein whereby the compositions are inconformance with the stability requirements of the present invention.Thus, when referring to a non-aqueous composition that is substantiallyfree of water, such composition is intended to include up to an amountof water whereby the composition conforms with the stabilityrequirements of the present invention. Preferably, the non-aqueousmetachromatic dye compositions of the present invention contain, atmost, trace amounts of water, and preferably do not include watertherein.

The non-aqueous metachromatic dye composition can be formulated using avariety of non-aqueous solvents in which the one or more metachromaticdyes intended to be included in the metachromatic dye composition aresoluble. Therefore, in order to determine whether a non-aqueous solventis a non-aqueous solvent or mixture of non-aqueous solvents that isutilizable in the non-aqueous metachromatic dye composition according tothe present invention, a non-aqueous solvent or mixture of thenon-aqueous solvents should be mixed with the one or more metachromaticdyes that are intended to be utilized in the non-aqueous metachromaticdye composition to determine if the one or more metachromatic dyes aresoluble in the non-aqueous solvent or mixture of non-aqueous solvents.If the one or more metachromatic dyes are soluble in the non-aqueoussolvent or mixture of non-aqueous solvents, the non-aqueousmetachromatic dye composition can be formulated with the non-aqueoussolvent or mixture of non-aqueous solvents and subjected to the hereindiscussed stabilization test to determine whether stabilization isachieved for the non-aqueous solvent or mixture of non-aqueous solvents.

The non-aqueous solvents can include, but are not limited to, alcoholssuch as methanol, ethanol, butanol, propanol, isopropanol, ethyleneglycol, propylene glycol, glycerin (glycerol); and organic solvents suchas methylcellosolve, hexane, pentane, heptane, toluene, xylene, benzene,dichlorobenzene, acetone, ethyl acetate, diethyl ether, acetonitrile,dimethylsulfoxide. Preferred solvents include methanol, ethanol,isopropanol, butanol, propanol and ethylene glycol.

Preferably, the non-aqueous solvent has a density at 25° C. of about0.95 to 1.2 g/cm³, more preferably 1 to 1.1 g/cm³, and even morepreferably 1 to 1.05 g/cm³, with a preferred value of density beingabout 1 g/cm³. To achieve this preferred density, the non-aqueoussolvent can be composed of one non-aqueous solvent that has a densitywithin the preferred range, or a mixture of non-aqueous solvents, withone, some or all of the non-aqueous solvents within the mixturecomprising a density that is not within the preferred density range;however, the density of the mixture of non-aqueous solvents willpreferably be within the preferred range.

Expanding upon the above, it is noted that single non-aqueous solvent ormixtures of non-aqueous solvents can be utilized in the non-aqueousmetachromatic dye compositions of the present invention. In this regard,it is noted that the viscosities of non-aqueous solvents can be lowerthan desired, whereby non-aqueous metachromatic dye compositionsincorporating these non-aqueous solvents can be too volatile. Stillfurther, it is noted that the viscosities of other non-aqueous solventscan be higher than desired, whereby non-aqueous metachromatic dyecompositions incorporating these non-aqueous solvents can be tooviscous. Therefore, it is preferable that the non-aqueous solvent have adensity at 25° C. of about 0.95 to 1.2 g/cm³, more preferably 1 to 1.1g/cm³, and even more preferably 1 to 1.05 g/cm³, with a preferred valuebeing about 1 g/cm³, or comprise mixtures of non-aqueous solvents, whichmixture has a density at 25° C. of about 0.95 to 1.2 g/cm³, morepreferably 1 to 1.1 g/cm³, and even more preferably 1 to 1.05 g/cm³,with a preferred value being about 1 g/cm³.

The mixture of non-aqueous solvents of differing densities can comprisevarious mixtures of non-aqueous solvents, with one preferred mixturebeing composed of methanol (density of 0.8 g/cm³ at 25° C.) and ethyleneglycol (density of 1.1 g/cm³ at 25° C.). Preferably, the higher densitynon-aqueous solvent is present in the mixture in a range of about 50 vol% to 95 vol %, with the lower density non-aqueous solvent being presentin the mixture in a range of about 5 vol % to 50 vol %. Particularlypreferred values include wherein the higher density non-aqueous solventis present in the mixture at about 60 vol %, with the lower densitynon-aqueous solvent being present in the mixture at about 40 vol %;wherein the higher density non-aqueous solvent is present in the mixtureat about 70 vol %, with the lower density non-aqueous solvent beingpresent in the mixture at about 30 vol %; and wherein the higher densitynon-aqueous solvent is present in the mixture at about 75 vol %, withthe lower density non-aqueous solvent being present in the mixture atabout 25 vol %. In other words, it is preferred that the volume ratio ofhigher density non-aqueous solvent to lower density non-aqueous solventis about 19:1 to 1:1, more preferably about 3:1 to 1.5:1, and even morepreferably about 3:1 to 2.3:1. A particularly preferred non-aqueoussolvent mixture comprises methanol present in an amount of about 25volume percent and ethylene glycol present in an amount of about 75volume percent.

Further beneficial improvements to the metachromatic dye compositionsaccording to the present invention can be achieved by utilizing highpurity materials, such as distilled water in the case of aqueousmetachromatic dye compositions, such as high purity non-aqueous solventsand high purity viscosity increasing agent or diluents in the case of anon-aqueous metachromatic dye composition.

In the case of non-aqueous solvents, high purity is referred to hereinas a purity of at least 99 wt %. It is noted that high purity is usuallyassociated with technical grade materials, as compared to reagent gradematerials.

The higher purity materials provide metachromatic dye compositionsaccording to the present invention with at least lower concentrations ofinterfering agents. For example, low purity materials can addinterfering agents, such as iron, calcium and/or magnesium to themetachromatic dye composition, which interfering agents can interferencewith both the metachromatic dye and the analytical test, such as ananalytical test for polyionic polymers.

Further beneficial improvements to the metachromatic dye compositionsaccording to the present invention can be achieved by storing themetachromatic dye composition in an oxygen free or substantially oxygenfree environment. The oxygen free or substantially oxygen freeenvironment can be obtained in any manner by which oxygen, such asmolecular oxygen, or oxidative components in the composition are avoidedand/or removed from the metachromatic dye composition. For example, andwithout limiting the present invention, an oxygen free or substantiallyoxygen free environment can be achieved by utilizing one or more of avariety of techniques including storing the metachromatic dyecomposition in a sealed container, purging the metachromatic dyecomposition with an inert gas, such as argon or nitrogen, utilizing anoxygen scavenger, such as, but not limited to, sodium sulfite, sodiumbisulfite, ascorbate, hydrazine, hydroquinone, benzohydroquinone, orsealing the metachromatic dye composition under vacuum or partialvacuum.

With regard to sealing the metachromatic dye composition in a container,it is noted that the metachromatic dye composition can be sealed in anycontainer that limits diffusion of oxygen into the metachromatic dyecomposition. Accordingly, the metachromatic dye composition can becontained in a container that is sealed, such as by utilizing a closureelement that can seal an opening in the container. For example, andwithout limiting the invention, the closure element can include a cap,such as a cap that can be screwed over the opening of the container toseal the opening, a cap that can be sealingly pressed against theopening to obtain a seal between the opening and the cap; or a cork typeelement, such as a rubber cork that can be inserted into the opening. Aparticularly preferred container is an 8 oz. Amber Boston Round, 24/400finish (referring to the threads on neck of bottles) including a 24/400black phenolic polyseal cone liner as a cap, as distributed by PENNBottle and Supply Co., Philadelphia, Pa., with the amber container beingmanufactured by Lawson Mardon Wheaton, Millbille, N.J., and the capbeing manufactured by Poly/Seal Corp., Baltimore, Md.

Still further, the containers can comprise completely sealed containers,such as ampoules. In such types of containers, it is often necessarythat the container be broken to enable release of the compositioncontained therein.

With respect to the use of oxygen scavengers, the oxygen scavenger cancomprise inorganic and/or organic materials, such as, but not limitedto, sodium sulfite, sodium bisulfite, ascorbate, hydrazine,hydroquinone, benzohydroquinone. The oxygen scavenger is preferablyadded to the metachromatic dye composition during its preparation, or assoon as possible after the metachromatic dye composition is prepared. Inthe instance where the oxygen scavenger is added after preparation ofthe metachromatic dye composition, it is preferred that the oxygenscavenger be added to the metachromatic dye composition immediatelyafter its production. Most preferably, the oxygen scavenger is added tothe solvent prior to the addition of the metachromatic dye.

With respect to purging with an inert gas, any purging technique can beutilized to remove oxygen from the metachromatic dye composition. Inthis regard, without limiting the invention, the inert gas, such asnitrogen and/or argon, can be caused to bubble through or pass over themetachromatic dye composition, preferably passed over the metachromaticdye composition. Of importance is that the purging technique issufficient to remove an effective amount of oxygen to enhance stabilityof the metachromatic dye composition. Moreover, the purging technique ispreferably adapted to remove as little of the solvent as possible,particularly in the case where the solvents are highly volatile. In oneexemplary purging technique, a piece of flexible tubing, such as Tygontubing, is connected to an inert gas tank, such as an argon tank, on oneend, and a glass or plastic pipette on the other end. The inert gas tankis turned on, and the gas is gently bubbled through the solution in acontainer, such as a beaker, an Erlenmeyer flask, etc.

Similarly, any technique for sealing a metachromatic dye compositionunder vacuum or partial vacuum can be utilized. Thus, for example, themetachromatic dye composition can be placed in a container, and a vacuumpulled on the container and its content to achieve a vacuum of less thanabout 100 mm of mercury (1 ppm O₂), more preferably less than leastabout 50 mm of mercury (0.5 ppm O₂), and even more preferably less thanabout 20 mm of mercury (0.2 ppm O₂).

Moreover, further beneficial improvements to the metachromatic dyecompositions according to the present invention can be achieved bystoring the metachromatic dye composition in a container that protectsthe metachromatic dye composition from light, especially ultravioletradiation. Thus, any container that at least partially limits, andpreferably completely blocks, the entrance of light into themetachromatic dye composition, can be preferably utilized to store themetachromatic dye composition. For example, the walls of the containercan be constructed from an opaque material that will completely blockthe entrance of light in the container and/or the container can beplaced in a dark environment, such as a cardboard carton or a styrenepackage. Still further, the walls of the container can be constructedfrom a translucent material that permits light to partially enter themetachromatic dye composition.

As examples of materials of construction for the container, withoutlimiting the invention, the container can be constructed of glass thatis treated and/or colored to prevent or limit penetration of light intothe container, such as amber colored glass, plastic containing materialsto block or limit the entrance of light, such as materials that will atleast limit or block the passage of ultraviolet radiation, or a metal. Apreferred amber bottle is obtainable from Nalgene International, and isconstructed from HDPE. An opaque container that prevents lighttransmission can be constructed of Teflon fluorinated ethylene propylene(FEP), which is used for extraordinary chemical resistance. Also a blackTeflon FEP container containing a carbon black pigment for zerotransmission of both visible and ultraviolet light is also availablefrom Nalgene International.

If the container is constructed from materials that may be aninterferant to the metachromatic dye or materials to be analyzed, it ispreferred to include a liner and/or a coating, such as a liner on a cap,or a coating on internal and/or external walls of the container. Inparticular, liners typically refer to inserts for the caps or closureson the bottles. For example, caps with flat, disc-type polyvinyl liners,caps with flat, disc-type Teflon TPE fluorocarbon resin liners,polypropylene film foam liners, black phenolic rubber-lined closures,and a preferred liner comprising black phenolic caps with corticallow-density polyethylene Poly-Seal liners that form especially goodseals. The caps or closures,can be constructed of the same materials asthe containers, for example, amber propylene or polypropylene screwclosures, polyethylene caps and/or Nalgene International TEFZEL®(ethylene-tetrafluoroethylene) closures. Coatings are typically used inreference to the outside of the container, and can include, for example,polyvinyl chloride, which is commonly used in coating acid bottles tohelp prevent breakage and spills. Moreover, a container, such as a clearcontainer, can be coated with aluminum foil, Styrofoam, etc.

Moreover, the container can be constructed from a material that istransparent to light if the container is stored in another containerthat blocks or limits the passage of light. For example, the material ofconstruction of the container can be clear glass or plastic, and, asdiscussed above, the container can be placed in a dark environment, suchas a cardboard carton or a styrene package, or have a liner positionedin the container and/or a coating placed exteriorly of the container toblock the transmission of light.

Materials of construction for a clear container can be glass and/or aresin, such as high density polyethylene (HDPE), low densitypolyethylene (LDPE), polycarbonate (PC), polyvinyl chloride (PVC),polypropylene (PP) and/or polymethylpentene (PMP). If the container isconstructed from these clear materials, preferably direct UV exposureshould be avoided, with the clear container being modified and/or placedin a dark environment. However, preferably, the container is constructedfrom a material that limits or blocks the passage of light into thecontainer.

Still further, beneficial improvements to the metachromatic dyecompositions according to the present invention can be achieved bystoring the metachromatic dye composition under low temperatureconditions. In particular, the metachromatic dye composition can bestored at temperature lower than about 15° C., more preferably lowerthan about 10° C., even more preferably lower than about 8° C. Themetachromatic dye composition is preferably stored at temperatures highenough so as not to freeze the metachromatic dye composition. Preferredtemperature ranges are about 15° C. to 3° C., more preferably about 10°C. to 4° C., more preferably about 8° C. to 4° C., and most preferablyabout 6° C. to 4° C.

It is noted that the herein described techniques for stabilization ofthe metachromatic dye compositions can be utilized in combination witheach other, and such combinations of techniques can provide enhancementof the stabilization. Thus, for example, a non-aqueous metachromatic dyecomposition can be stored in a container that limits or blocks lightpenetration into the bottle, with or without any combination oftechniques for removal of oxygen from the non-aqueous metachromatic dyecomposition, such as any combination of purging and/or use of oxygenscavengers, with or without lower storage temperatures. Moreover, theaqueous metachromatic dye composition can be adjusted to an alkaline pHwith or without storage under dark conditions, and with or withoutstorage at lower temperatures.

The concentration of the metachromatic dye in the solvent is preferablya concentration of the metachromatic dye that is in excess of that whichis expected to react with the analyte to be determined, such as apolyionic polymer. For example, for the polyionic polymer, HPS-I,obtained from BetzDearborn, Inc., Trevose, Pa, at a concentration ofabout 0.5 ppm of polymer in the sample, the metachromatic dyecomposition preferably has a metachromatic dye molar concentration of atleast about 9×10⁻⁵M.

It is noted that one mole of metachromatic dye reacts with 0.05 molesSO₄ ⁻ (mole ratio of 1:0.05), or 0.15 moles of COO⁻ (mole ratio of1:0.15), or one mole of metachromatic dye to 10⁻³ moles HPS-I (moleratio of 1:0.001). Thus, the mole ratio of metachromatic dye moleculesto SO₄ ⁻ ranges from about 0.05 to 100, and the mole ratio ofmetachromatic dye molecules to COO⁻ ranges from about 0.15 to 100. It istherefore preferred that the mole ratio of metachromatic dye topolycarboxylate and/or sulfonate groups is about 0.1 to 100, morepreferably about 1 to 50, and even more preferably about 1 to 30.

The concentration of the metachromatic dye in the metachromatic dyecomposition is optimized so the maximum metachromatic absorbance isobtained for a polyionic material to be assayed by the followingtechnique. Different concentrations of metachromatic dye composition arereacted with known concentrations of the polyionic material to beassayed, such as HPS-I, and a plot of absorbance vs. concentration aspolyionic material is plotted. The slope of the curve is determined, andthe optimum metachromatic dye composition includes a concentration ofmetachromatic dye, or a concentration range of metachromatic dye thatprovides the largest slope with a small or smallest intercept. In thismanner, the metachromatic dye composition is optimized so that a smallchange in the concentration of the polyionic material to be assayedcorresponds to a large change in absorbance over the operable absorbancerange of the metachromatic dye.

A particularly preferred metachromatic dye composition is formulated bymixing 0.0844 g of metachromatic dye, e.g., pinacyanol chloride, with250 mL of non-aqueous solvent, e.g., methanol, and adding the resultingmixture to 750 mL of viscosity increasing agent, e.g., ethylene glycol,and mixing for a sufficient amount of time to homogenize the solution,such as 30 minutes. This preferably provides a preferred composition of25 vol % methanol and 75 vol % ethylene glycol with 0.0844 grams ofpinacyanol chloride.

With respect to the formulation of the metachromatic dye compositions,in the case of a non-aqueous solvent system, the dye can be mixed withone or more solvents and/or one or more viscosity increasing agents.When mixed with plural solvent/viscosity materials, the metachromaticdye can be individually mixed with one or more of the materials, andsubsequently mixed with any other materials in any order, or can bemixed with the total combination of materials. However, it is preferredto mix the metachromatic dye with one of the more soluble materials,most preferably the most soluble material, and then mix the resultingcomposition with the other solvent materials.

When mixed with solvents that do not as easily solubilize themetachromatic dye, it is preferred that the resulting composition bemixed for a sufficient period of time so that the solution is clear inthat floating matter is not visible. For example, the mixing could beperformed for, but not limited to, about 30 minutes, to homogenize themetachromatic dye in the solvent system.

Preferably, a dye such as pinacyanol chloride is dissolved in anoxygen-free solvent and is packaged in glass (amber) ampoules. Thepackaging may be sealed under vacuum for maximum stability. Storageunder these conditions provides a dye with suitable stability bypreventing oxygen and/or light from contacting the dye solution.

The metachromatic dye compositions of the present invention can beutilized in the determination of polyionic substances, particularly,polyionic polymers, in various environments, including, but not limitedto, in aqueous environments, such as potable water, industrial systems,cooling waters, boiler systems, industrial processes, and water andwaste water applications. For example, the metachromatic dyecompositions can be utilized to determine polycarbonates and sulfonatesdisclosed in U.S. Pat. No. 4,894,346 to Myers, which is incorporated byreference hereto in its entirety. Moreover, the metachromatic dyecompositions of the present invention can be utilized to monitor polymerconcentrations in cooling water field samples, such as to monitor HPS-I(acrylic acid/1-allyloxy, 2-hydroxypropylsulfonate), such as disclosedin U.S. Pat. No. 4,659,481 to Chen, which is incorporated by referencehereto in its entirety, as well as PESA (polyepoxysuccinic acid disodiumsalt), such as disclosed in U.S. Pat. No. 5,062,962 to Brown et al.,which is incorporated by reference hereto in its entirety.

To be utilizable as a metachromatic dye for analytical purposes, themetachromatic dye should be able to pass a two part test. In the firsttest, which is a quality of the metachromatic dye test, themetachromatic dye is mixed one part by volume with 50 parts of distilledwater. The resulting dye solution should provide a visible absorbancewithin 1.000±0.100 AU (Absorbance Units) utilizing a 1 inch (2.54 cm)path length when measured in a spectrophotometer at 600 nm, threeminutes after production of the metachromatic dye solution.

If a metachromatic dye passes the first test, it is subjected to thesecond test. In the second test, a 1 ppm solution of polymer indistilled water is prepared by mixing 100 ml of 1 ppm standard solutionof polymer in distilled water 2 ml of buffer, preferably meta buffer(10.1 wt % EDTA (ethylendiaminetetraacetic acid)tetrasodium salt, 11 wt% potassium I 0 phosphate monobasic, and 78.9 wt % distilled water) and5 ml of metachromatic dye. The absorbance of this solution is measuredand compared to a standard calibration curve utilizing a standard 1 ppmsolution of polymer in distilled water, such as illustrated in FIG. 1and tabulated in Table 3. The measured absorbance is read against thecalibration curve to determine the concentration of polymer. Theconcentration of polymer should be between 90% and 110% of the 1 ppmconcentration. For example, in the illustrated embodiment of FIG. 1 andTable 3, the concentration should be between 0.9 and 1.1 for themeasured absorbance for the metachromatic dye to pass the second test.

The determination of polyionic substances can be performed utilizingvarious techniques, and the following non-limiting techniques aredescribed to provide examples of both off-line and on-line methods ofperforming the determination. For example, the off-line determination ofHPS-I can be accomplished by diluting the sample to obtain a 0.1 to 1.5ppm concentration of HPS-I, of which 100 ml is mixed with 2 ml of abuffer, such as meta buffer, and 5 ml of metachromatic dye composition.The mixture is preferably swirled for about 10 seconds, and at 45 to 50seconds, 25 ml is preferably transferred to the sample cell. At as closeto possible to the desired test time, such as 60 seconds, the absorbancemeasurement is performed, such as preferably at 480 nm in the case ofHPS-I.

It is noted that glassware and sample cell are preferably rinsed withmethanol after each use to avoid dye staining, then thoroughly rinsedwith distilled water. Moreover, the use of disposable pipettes ispreferred, as it is hard to clean and reuse pipettes.

The on-line determination of polyionic substances, such as HPS-I, can beachieved utilizing a ChemScan Analyzer, Model UV-6101, manufactured byApplied Spectrometry Associates, Inc. (ASA), Waukesha, Wis. 53186. Thismodel allows the user to program a “read” sequence that can include upto twenty different mechanical, optical and mathematical functions. Thesample volume is preferably about 10 ml, and the pathlength ispreferably 0.5 inch (1.27 cm). In the test, the flow cell is flushed andfilled with a sample and the cell is air purged for 5 seconds to createa small headroom for reagent additions. A buffer such as 0.5 ml of metabuffer is injected and mixed for 5 seconds before injecting 1 ml of thedye. The dye is mixed for 5 seconds and a visible absorbance scanbetween 400 and 650 nm is made after 20 seconds of reaction time. Amulti-wavelength chemometric calibration is applied to the spectrum todetermine the polymer concentration in the sample.

The invention will now be described with respect to certain exampleswhich are merely representative of the invention and should not beconstrued as limiting thereof.

EXAMPLES

The invention is illustrated in the following non-limiting examples,which are provided for the purpose of representation, and are not to beconstrued as limiting the scope of the invention. All parts andpercentages in the examples are by weight unless indicated otherwise.

Examples 1-8

The stability of pinacyanol chloride, obtained from Aldrich ChemicalCo., Milwaukee, Wis., in different solvent systems is tested, with allconditions being at room temperature, using a 1 inch (2.54 cm)pathlength cuvette, by dissolving the pinacyanol chloride in eachsolvent system, and recording the absorbance at 600 nm in a DR2000/2010Spectrophotometer supplied by HACH Company, Loveland, Colo., after threeminutes mixing time. It is noted that where less than 100 vol %indicated for a solvent system, the balance was distilled water.Moreover, solubility issues led to the use of different concentrationsof the dye in the different solutions. The concentrations and resultsare illustrated in Table 1, with storage of the dye solutions in amberbottles being in air for the time indicated in Table 1. Each absorbancewas measured for a minimum of 6 samples, and the average value isillustrated in Table 1. The % change from day 1 shown in the Table 1,and the other tables which follow is the change of the final measuredvalue as compared to the initial measurement.

The results illustrated in Table 1 demonstrate that Examples 2 (100 vol% ethanol), 3 (50 vol % ethylene glycol in distilled water), 5 (100 vol% methanol) and 8 (100 vol % Methyl Cellosolve) produced the most stabledye solution.

TABLE 1 Stability of Pinacyanol Chloride in Solvent Systems as afunction of time in days Ex. No. 1 2 3 4 5 6 7 8 Solvent DistilledEthanol Ethylene Methanol Methanol Methyl Methyl Methyl Water (100%)Glycol (5%) (100%) Cellosolve Cellosolve Cellosolve (50%) (2%) (5%)(100%) Dye 8.84 × 1.1 × 9.2 × 8.93 × 1.79 × 9.0 × 9.0 × 1.8 × Conc. 10⁻⁵M 10⁻³ M 10⁻⁵ M 10⁻⁵ M 10⁻³ M 10⁻⁵ M 10⁻⁵ M 10⁻³ M Time (days)Absorbance at 600 nm 1 1.370 1.058 1.437 1.446 1.446 1.445 1.445 1.445 21.318 3 1.289 4 1.250 5 1.242 6 1.229 1.418 1.364 1.365 1.419 20  1.42135  1.428 58  1.464 82  1.057 % Change −9.3 −0.1 −0.6 −15.0 1.2 −5.6−5.5 −1.7 from day 1

Examples 9-11

0.0844 g pinacyanol chloride is dissolved in a solvent system of 250milliliters (ml) of technical grade methanol obtained from CoyneChemical,, Croydon, Pa., and 750 ml of ethylene glycol, obtained fromFischer Scientific, Pittsburgh, Pa. (Laboratory Grade indicated to betypically>99% pure), for a total volume of 1 liter. The solutions werestored at three temperatures of 4° C., 20° C. and 40° C. for the timeperiods indicated in Table 2. Storage of the samples and the absorbancemeasurements were made in a similar manner as set forth in Example 1except all experiments were run a minimum of three times with theaverage shown in Table 2. These examples show that metachromatic dyecompositions according to the present invention are stable for lengthyperiods, including about 7.5 months.

TABLE 2 Stability of Pinacyanol Chloride in 25 vol %/75 vol %Methanol/Ethylene Glycol (1:3) Stored at Different Temperatures Ex. No.9 10 11 Temperature 40° C. 20° C. 4° C. Time (days) Absorbance at 600 nm 1 1.011 1.011 1.011  48 1.025 1.040 1.014  55 1.021 1.028 1.051  831.079 1,090 1.087 123 0.948 0.889 0.973 166 1.027 0.995 1.083 194 0.9890.989 1.094 230 0.977 0.942 1.077 % Change −3.4   −6.8   6.5  from day 1

Example 12

A calibration curve for HPS-I, obtained from BetzDearborn Division ofHercules Incorporated, Trevose, Pa., was prepared by reacting differentconcentrations of the HPS-I dissolved in distilled water mixed with 2 mlof META buffer and 5 ml of the pinacyanol chloride solution inmethanol/ethylene glycol of Examples 9-11 except that the methanol isCertified A.C.S. Grade Methanol with a purity of 99.8% obtained fromFischer Scientific, Pittsburgh, Pa. The visible absorbance at 480 nm wasmeasured after reacting for 1 minute using a DR2000/2010Spectrophotometer available from HACH Company, Loveland, Colo. A 1-inch(2.54 pathlength cuvette holding a total volume of 25 ml was utilized,and the dye solutions were stored in amber bottles in air for theduration of all experiments. Each absorbance was measured for a minimumof 3 samples, and the average value is illustrated in Table 3, and theresulting calibration curve is shown in FIG. 1.

TABLE 3 Concentration (ppm) of HPS-I Absorbance @ 480 nm 0 0.157 0.10.213 0.2 0.267 0.5 0.403 1.0 0.582 1.5 0.696

Examples 13-27

The pinacyanol chloride solution in a 25:75 vol % of methanol toethylene glycol of Examples 9-11 was stored for varying period of times.The stored pinacyanol chloride solutions were prepared as in Example 12,and then reacted with the same known concentration of HPS-I, and thevisible absorbance was measured in the same manner as in Example 12.

The solution having a known concentration of HPS-I was prepared bymixing HPS-I in distilled water to give a concentration of 1 ppm HPS-1to achieve a test solution that provides an absorbance reading withinthe calibration range of 0-1.5 ppm, as illustrated in FIG. 1. Theresults are shown in Table 4.

TABLE 4 Concentration of HPS-I Recovered as a Function of Time (in days)Using Pinacyanol Chloride in 25 Vol % MeOH:75 Vol % Ethylene Glycol.Time Temperature Absorbance Concentration Example No. (Days) (° C.) (@480 nm) Recovered, ppm 13  83 40 0.605 1.07 14  83 20 .0594 1.03 15  83 4 0.607 1.07 16 123 40 0.591 1.02 17 123 20 0.584 1.00 18 123  4 0.5881.01 19 166 40 0.593 1.03 20 166 20 0.579 0.99 21 166  4 0.603 1.07 22194 40 0.594 1.03 23 194 20 0.575 0.97 24 194  4 0.604 1.07 25 230 400.590 1.01 26 230 20 0.572 0.96 27 230  4 0.595 1.03

Examples 28-56

The stability of 9×10⁻⁵ pinacyanol chloride, obtained from AldrichChemical Co., Milwaukee, Wis., is tested in aqueous media as shown inTables 5-14, with all conditions being at room temperature, using a 1inch (2.54 cm) pathlength cuvette, by dissolving the pinacyanol chloridein an aqueous system, and recording the absorbance at 600 nm in theabove-noted DR2000/2010 Spectrophotometer after a 3 minute mixing time.Storage of the dye solutions is in amber bottles in air for the timeindicated in the tables. Each absorbance was measured in at leastduplicate, and the average value is illustrated in the tables.

Table 5 shows the stability of pinacyanol chloride in the presence ofstrong base.

Table 6 shows the stability of pinacyanol chloride in the presence ofoxygen scavenger.

Table 7 shows the stability of pinacyanol chloride in the presence ofoxygen scavenger.

Table 8 shows the stability of pinacyanol chloride in the presence ofstrong based and oxygen scavenger.

Table 9 shows the stability of pinacyanol chloride in the presence ofoxygen scavenger.

Table 10 shows the stability of pinacyanol chloride in the presence ofoxygen scavenger.

Table 11 shows the stability of pinacyanol chloride in the presence ofstrong base and oxygen scavenger.

Table 12 shows the stability of pinacyanol chloride in the presence ofbuffer.

Table 13 shows the stability of pinacyanol chloride in the presence ofstrong acid.

Table 14 shows the stability of pinacyanol chloride in the presence ofoxygen scavenger.

As can be seen from the results depicted in these Tables 5-14, thepresence of a strong acid or oxygen scavenger per se, the dye discolorsquickly. In the presence of 0.15 wt/wt of NaOH, the dye fading isminimal (˜3% in 6 days).

TABLE 5 Stability of Pinacyanol Chloride in the Presence of Strong BaseTime Absorbance % change Ex. No. (days) System @ 600 nm from Day 1Observations 28 1 Distilled Water (DI) + 1.196 Dark Purple, 1 ml NaOH(1N) No Change 29 2 DI + 1.213 1.4 Dark Purple, 1 ml NaOH (1N) No Change30 6 DI + 1.164 −2.7 Dark Purple, 1 ml NaOH (1N) No Change

TABLE 6 Stability of Pinacyanol Chloride in the Presence of OxygenScavenger Time Absorbance % change Ex. No. (days) System @ 600 nm fromDay 1 Observations 31 1 DI + 0.1 gm Na₂SO₃ 1.089 Sky Blue, SignificantColor Change 32 2 DI + 0.1 gm Na₂SO₃ 1.14  4.7 Sky Blue, SignificantColor Change 33 6 DI + 0.1 gm Na₂SO₃ 0.741 −32 Sky Blue, SignificantColor Change

TABLE 7 Stability of Pinacyanol Chloride in the Presence of OxygenScavenger Time Absorbance % change Ex. No. (days) System @ 600 nm fromDay 1 Observations 34 1 DI + 0.2 gm Na₂SO₃ 1.03 Sky Blue, SignificantColor Change 35 2 DI + 0.2 gm Na₂SO₃ 0.12 −88.3 Sky Blue, SignificantColor Change 36 6 DI + 0.2 gm Na₂SO₃  0.557 −44 Precipitation

TABLE 8 Stability of Pinacyanol Chloride in the Presence of Strong Baseand Oxygen Scavenger Time Absorbance % change Ex. No. (days) System @600 nm from Day 1 Observations 37 1 DI + 0.1 gm Na₂SO₃ + 1.07  Sky Blue,1 mL NaOH (1N) Significant Color Change 38 2 DI + 0.1 gm Na₂SO₃ + 1.53443.4 Sky Blue, 1 mL NaOH (1N) Significant Color Change 39 6 DI + 0.1 gmNa₂SO₃ + 0.858 −19.8 Sky Blue, 1 mL NaOH (1N) Significant Color Change

TABLE 9 Stability of Pinacyanol Chloride in the Presence of OxygenScavenger Time Absorbance % change Ex. No. (days) System @ 600 nm fromDay 1 Observations 40 1 DI + 0.1 gm NaNO₂ 0.884 Sky Blue, SignificantColor Change 41 2 DI + 0.1 gm NaNO₂ 0.719 −18.7 Sky Blue, SignificantColor Change 42 6 DI + 0.1 gm NaNO₂ 0.713 −19.3 Precipitation

TABLE 10 Stability of Pinacyanol Chloride in the Presence of OxygenScavenger Time Absorbance % change Ex. No. (days) System @ 600 nm fromDay 1 Observations 43 1 DI + 0.2 gm NaNO₂ 1.16  Sky Blue, SignificantColor Change 44 2 DI + 0.2 gm NaNO₂ 1.117 −3.7 Sky Blue, SignificantColor Change 45 6 DI + 0.2 gm NaNO₂ 0.871 −25 Precipitation

TABLE 11 Stability of Pinacyanol Chloride in the Presence of Strong BaseOxygen Scavenger Time Absorbance % change Ex. No. (days) System @ 600 nmfrom Day 1 Observations 46 1 DI + 0.1 gm NaNO₂ + 0.893 Sky Blue, 1 mLNaOH (1N) Significant Color Change 47 2 DI + 0.1 gm NaNO₂ + 0.908 1.7Sky Blue, 1 mL NaOH (1N) Significant Color Change 48 6 DI + 0.1 gmNaNO₂ + 1.020 14 Sky Blue, 1 mL NaOH (1N) Significant Color Change

TABLE 12 Stability of Pinacyanol Chloride in the Presence of Buffer Ex.Time Absorbance % change No. (days) System¹ @ 600 nm from Day 1Observations 49 1 Meta Buffer 1.038 Dark Purple, No Change 50 2 MetaBuffer 1.701 63.9 Precipitation 51 6 Meta Buffer 0.923 −11 Precipitation¹Meta buffer includes 10.1% EDTA (Ethylendiaminetetraacetic acid)Tetrasodium Salt, 11% Potassium Phosphate Monobasic, and 78.9% DistilledWater.

TABLE 13 Stability of Pinacyanol Chloride in the Presence of Strong AcidEx. Time Absorbance % change No. (days) System @ 600 nm from Day 1Observations 52 1 DI + 2 drops Colorless H₂SO₄ (10N) 53 2 DI + 2 dropsColorless H₂SO₄ (10N) 54 6 DI + 2 drops Colorless H₂SO₄ (10N)

TABLE 14 Stability of Pinacyanol Chloride in the Presence of OxygenScavenger Ex. Time Absorbance % change No. (days) System @ 600 nm fromDay 1 Observations 55 1 DI + 0.1 gm 1.287 hydro- quinone 56 6 DI + 0.1gm Light hydro- Brown, quinone Significant Color Change

Examples 57-61

The stability of pinacyanol chloride, obtained from Aldrich ChemicalCo., Milwaukee, Wis., in different solvent systems is tested, with allconditions being at room temperature, using a 1 inch (2.54 cm)pathlength cuvette, by dissolving the pinacyanol chloride in eachsolvent system, and recording the absorbance at 600 nm in a DR2000/2010Spectrophotometer supplied by HACH Company, Loveland, Colo., after threeminutes mixing time. It is noted that where less than 100 vol %indicated for a solvent system, the balance was distilled water.Moreover, solubility issues led to the use of different concentrationsof the dye in the different solutions. Storage of the dye solutions isin amber bottles in air at 4° C. for the time indicated in the tables.Each absorbance was measured for a minimum of 6 samples, and the averagevalue is illustrated in the Table 15.

TABLE 15 Stability of Pinacyanol Chloride in Solvent Systems as afunction of time in days at 4° C. Ex. No. 57 58 59 60 61 SolventMethanol Methanol Methyl Methyl Methyl (5 vol %) (100 CellosolveCellosolve Cellosolve vol %) (2 vol %) (5 vol %) (100 vol %) Dye Conc.8.93 × 1.79 × 9.0 × 9.0 × 1.8 × 10⁻⁵ M 10⁻³ M 10⁻⁵ M 10⁻⁵ M 10⁻³ M Time(days) Absorbance at 600 nm 1 1.489 1.469 1.439 1.445 1.445 6 1.4261.462 1.364 1.365 1.419 20  1.421 22  1.446 % Change −4.2 −1.6 −5.2 −5.5−1.7 from day 1

Although data shows promising results, it is noted that storage of themetachromatic dye compositions under refrigeration in the field is notas desirable as storage under ambient conditions.

Examples 62-63

The stability of pinacyanol chloride, obtained from Aldrich ChemicalCo., Milwaukee, Wis., in distilled water, with purging with N₂ gas inroom temperature (20° C.) and low temperature, i.e., 4° C., studiesutilizing conditions as set forth in Examples 57-61. The results areshown in Table 16

TABLE 16 Stability of Pinacyanol Chloride in Distilled Water UsingNitrogen Purge At Room Temperature and 4° C. Ex. No. 62 63 (20° C.) (4°C.) Time (days) Absorbance at 600 nm 1 1.463 1.463 7 1.392 1.466 23 1.446 % Change −4.8 −1.2 from day 1

Although data shows promising results, it is noted that storage of themetachromatic dye compositions under refrigeration in the field is notas desirable as storage under ambient conditions. Moreover,stabilization by purging the metachromatic dye composition after eachuse is not as desirable as stabilization with a technique that does notrequire repeated efforts on the part of field personnel.

Example 64

Varying concentrations of pinacyanol chloride, obtained from AldrichChemical Co., Milwaukee, Wis., in a solution of 25 vol % methanol,Certified A.C.S. Grade Methanol with a purity of 99.8% obtained fromFischer Scientific, Pittsburgh, Pa., to 75 vol % ethylene glycol,obtained from Fischer Scientific, Pittsburgh, Pa. (Laboratory Gradeindicated to be typically>99%, are prepared, and reacted with 0.2 mg/Land 1.0 mg/L HPS-I, obtained from BetzDearborn Division of HerculesIncorporated, Trevose, Pa., with the visible absorbance being measuredin the same manner as in Example 12. The results are illustrated inTable 17.

TABLE 17 Conc (M) Absorbance Absorbance Pinacyanol Chloride in @ 480 nm@ 480 nm 25% Methanol:75% Ethylene Glycol 0.2 mg/L HPS-I 1.0 mg/L HPS-ILinear Equation 1.07 × 10⁻⁴ 0.175 0.451 y = 0.345x + 0.106 1.43 × 10⁻⁴0.236 0.487 y = 0.3138x + 0.1733 2.14 × 10⁻⁴ 0.309 0.604 y = 0.3688x +0.2353 2.17 × 10⁻⁴ 0.318 0.621 y = 0.3788x + 0.2423 2.91 × 10⁻⁴ 0.4310.719 y = 0.36x + 0.359 3.58 × 10⁻⁴ 0.447 0.810 y = 0.4538x + 0.3563

A plot of absorbance vs. concentration is plotted, as illustrated inFIG. 2. It is noted that as the concentration of the dye solutionincreases, the curves in FIG. 2 decrease. The slope of the curve isdetermined, and the optimum metachromatic dye concentration provides asmall change in the concentration of the HPS-I providing a large changein absorbance. This concentration provides a compromise between thesteepest slope and the smallest intercept, so that a small change in theconcentration of HPS-I in a water sample will correspond to a largechange in absorbance. Thus, a molar concentration of 2.17×10⁻⁴ is seento be a preferred concentration of the pinacyanol chloride. Inparticular, a dye concentration of 2.17×10⁻⁴M not only gives the highestsensitivity (as seen by the increase in the slope on the calibrationcurve in FIG. 2) but also produces the best accuracy in the calibrationrange (0.3 to 0.6 absorbance units for the recommended spectral rangefor a HACH DR2000/2010).

Example 65

The stability of metachromatic dye compositions in sealed glass ampouleswas tested utilizing water purged with argon as follows.

The water used is distilled water which is purged thoroughly with argonto remove dissolved oxygen.

Three metachromatic dye compositions are prepared including ametachromatic dye concentration of 35 mg/L (9.0×10⁻⁵M) in the followingthree solvent systems:

a. 100% aqueous solution

b. 50% v/v water/propylene glycol

c. 50% v/v water/ethylene glycol.

The solutions were packaged in AccuVac ampoules, obtained from HachCompany, Loveland, Colo., and Voluette ampoules, obtained from HachCompany, Loveland, Colo., immediately after preparation. AccuVacampoules were filled with 1 mL of the dye solution, and the total volumeof the ampoule is 13.5 mL. The Voluette ampoules are filled with 2 mL ofthe dye solution, and the total volume of the ampoule is 2.2 mL.

The Voluette ampoules are amber in color. The AccuVac ampoules areplaced into styrofoam containers as soon as they were prepared toprotect them from light.

The packaging of the Voluette is under atmospheric pressure, whereas theAccuVac is under vacuum.

Results of the tests are shown in Table 18 for the metachromatic dyecompositions, and in Table 19 for reaction with a HPS-I standardsolution.

With regard to testing the AccuVac ampoules, it is noted that a singleAccuVac ampoule gives vastly different absorbances depending on theorientation in the cell holder. Absorbances varied from 0.307 to 0.358AU or 0.05 AU for the test which is ˜15% error. The reasons for thesesignificant variances include that the ampoules did not have aconsistent diameter, the ampoules were becoming stained with the dye,i.e., the dye precipitated, and imperfections in the quality of theglass.

The dye in the Voluette ampoules continued to show stability after 2.5months time.

TABLE 18 Quality Check (@ 600 nm) of dye solutions (25 mL of DI water +2 mL of dye solution) Solvent H₂O/Ethylene H₂O/Propylene H₂O/EthyleneH₂O/Propylene H₂O Glycol Glycol Glycol Glycol AccuVac AccuVac AccuVacVoluette Voluette Time (Days) Absorbance @ 600 nm 1 1.587 1.722 1.7821.736 1.707 4 1.301 1.479 1.519 1.731 1.691 6 1.147 1.400 1.414 1.6331.664 20  1.622 1.674 82  1.625 1.663 % Change −28 −19 −21 −6.4 −2.6from day 1

TABLE 19 Quality Check (@ 480 nm) of 25 mL of a 0.5 ppm HPS-I standardsolution + 1 mL Meta Buffer + 2 mL of dye solution H₂O/EthyleneH₂O/Propylene H₂O/Ethylene H₂O/Propylene Glycol Glycol Glycol GlycolTime AccuVac AccuVac Voluette Voluette  6 0.345 0.328 0.343 0.334 820.332 0.328

It is noted that the foregoing examples have been provided merely forthe purpose of explanation and are in no way to be construed as limitingof the present invention. While the present invention has been describedwith reference to an exemplary embodiment, it is understood that thewords which have been used herein are words of description andillustration, rather than words of limitation. Changes may be made,within the purview of the appended claims, as presently stated and asamended, without departing from the scope and spirit of the presentinvention in its aspects. Although the present invention has beendescribed herein with reference to particular means, materials andembodiments, the present invention is not intended to be limited to theparticulars disclosed herein; rather, the present invention extends toall functionally equivalent structures, methods and uses, such as arewithin the scope of the appended claims.

What is claimed is:
 1. A process for producing a stabilized aqueoussolution of metachromatic dye, comprising forming a solution ofmetachromatic dye in aqueous solvent, the metachromatic dye comprisingpinacyanol chloride, and the solution having a metachromatic dyestabilizing pH, and the stabilized aqueous solution of metachromic dyehaving a percent change in absorbance of less than about 10% when storedfor a period of about one week.
 2. The process according to claim 1wherein the solution has a pH of at least about
 8. 3. The processaccording to claim 2 wherein the solution has a pH of at least about 10.4. The process according to claim 3 wherein the solution has a pH of atleast about
 11. 5. The process according to claim 1 wherein the solutionhas a pH of about 8 to
 14. 6. The process according to claim 5 whereinthe solution has a pH of about 11 to
 12. 7. The process according toclaim 6 wherein the solution has a pH of about 11 to 11.5.
 8. Theprocess according to claim 1 including combining at least one basicmaterial with the solution.
 9. The process according to claim 8 whereinthe at least one basic material comprises a buffer.
 10. The processaccording to claim 8 wherein the at least one basic material comprisesat least one of sodium hydroxide, potassium hydroxide and lithiumhydroxide.
 11. The process according to claim 10 wherein said at leastone basic material comprises sodium hydroxide.
 12. The process accordingto claim 1 including combining at least one oxygen scavenger with thesolution.
 13. The process according to claim 1 wherein the solution issubstantially free of oxygen.
 14. A process for producing ametachromatic dye solution for analytical determination of at least onepolyionic substance, comprising combining metachromatic dye andnon-aqueous solvent so as to form a metachromatic dye solution which issubstantially free of water, the metachromatic dye comprising pinacyanolchloride, the metachromatic dye solution including a concentration ofthe metachromatic dye which provides maximum metachromatic absorbancefor the at least one polyionic substance when the at least one polyionicsubstance is present at a concentration of 0.1 to 1.5 ppm, and thenon-aqueous solvent comprises at least one of methanol, ethanol,butanol, isopropanol, propanol, ethylene glycol, methylcellosolve,hexane, pentane, heptane, toluene, xylene, benzene, dichlorobenzene,acetone, ethyl acetate, diethyl ether, acetonitrile anddimethylsulfoxide.
 15. The process according to claim 14 wherein thenon-aqueous solvent comprises at least one of methylcellosolve, hexane,pentane, heptane, toluene, xylene, benzene, dichlorobenzene, acetone,ethyl acetate, diethyl ether, acetonitrile, dimethylsulfoxide.
 16. Theprocess according to claim 14 wherein the non-aqueous solvent comprisesat least one of methanol, ethanol, butanol, isopropanol, propanol andethylene glycol.
 17. The process according to claim 14 wherein thenon-aqueous solvent has a density at 25° C. of about 0.95 to 1.2 g/cm³.18. The process according to claim 17 wherein the non-aqueous solventhas a density at 25° C. of about 1 to 1.1 g/cm³.
 19. The processaccording to claim 18 wherein the non-aqueous solvent has a density at25° C. of about 1 to 1.05 g/cm³.
 20. A process for producing ametachromatic dye solution, comprising combining metachromatic dye andnon-aqueous solvent so as to form a metachromatic dye solution which issubstantially free of water, the metachromatic dye comprising pinacyanolchloride, the non-aqueous solvent having a density at 25° C. of about0.95 to 1.2 g/cm³ and comprising a mixture of methanol and ethyleneglycol.